Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry...

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Analysis and Sorting Analysis and Sorting of T reg Cells on the of T reg Cells on the FACSAria FACSAria John Daley John Daley Director of Flow Cytometry Director of Flow Cytometry Hematologic Neoplasia Hematologic Neoplasia Dana-Farber Cancer Institute Dana-Farber Cancer Institute Harvard Medical School Harvard Medical School tp://www.bdbiosciences.com/features/products Picture credit:
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Transcript of Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry...

Page 1: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Analysis and Sorting of T reg Analysis and Sorting of T reg Cells on the FACSAriaCells on the FACSAria

John DaleyJohn DaleyDirector of Flow CytometryDirector of Flow Cytometry

Hematologic NeoplasiaHematologic NeoplasiaDana-Farber Cancer InstituteDana-Farber Cancer Institute

Harvard Medical SchoolHarvard Medical School

http://www.bdbiosciences.com/features/products

Picture credit:

Page 2: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

http://www.bdbiosciences.com/features/products/display_product.php?keyID=162

From BD Biosciences WebsiteFeatured products:Regulatory T cells

Page 3: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

The way we used to sort Human The way we used to sort Human CD4+CD25+ PBL’s on the CD4+CD25+ PBL’s on the

FACSAriaFACSAria

SORT TOP2- 3% CD25 HI

Page 4: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Then CD45Ra was added to the Then CD45Ra was added to the Sort DesignSort Design

Page 5: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Four way reg sort

Multicolor(5) Minor populations : Four way sorting: Treg Story

CD45ra FITCCD127 PECD25 PE-CY5CD4 PE-CY7CD3 PAC BLUE

Page 6: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Compensation ComparisonsCompensation Comparisons

Manual Comp with cells and actual Manual Comp with cells and actual reagentsreagents

Autocomp with cells and actual reagentsAutocomp with cells and actual reagents

Autocomp with beads and actual reagentsAutocomp with beads and actual reagents

Autocomp with beads and CD45+ Autocomp with beads and CD45+ reagentsreagents

Autocomp with cells and CD45+ reagentsAutocomp with cells and CD45+ reagents

Page 7: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

BD cells Cell Autocomp

BD cellsBead AutoComp

Page 8: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Treg Post Sort Reanalysis

Page 9: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

99.4 97.198.4 99.9

POST SORT REANALYSIS: HUMAN PBL CD3+CD4+ CD45RA+ &CD45RA- Treg+/Teffector+/ --

Page 10: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Actual reagents cells compensation

cells

beads

Actual reagents beads compensation

cells beads

Page 11: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

FoxP3 Expression in CD4+ Treg FoxP3 Expression in CD4+ Treg CD45RA+CD45RA+

Page 12: Analysis and Sorting of T reg Cells on the FACSAria John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical.

Fluorescence minus 1Fluorescence minus 1